Method of producing glucuronic acid



Patented Aug. 29, 1950 OFFICE METHOD OF PRODUCING GLUCURONIC ACID Earl A. Peterman, Detroit, Mich.

No Drawing. Application May 28, 1947, Serial No. 751,141

6 Claims.

The present invention relates to methods of synthesizing glucuronic acid and salts and compounds thereof, and constitutesan improvement over the processes for the same purposes disclosed in applicants copending application, Serial No. 629,315, filed November 17, 1945.

In order to synthesize glucuronic acid, it is necessary to oxidize the free primary alcohol (hydroxyl) group on aglucose molecule without breaking the molecule or oxidizing the aldehyde group. Such selective oxidation can be accomplished by coupling the glucose molecule at its aldehyde end with a substance which will protect that end of the molecule from oxidation. Thereafter, the alcohol group on the molecule may be oxidized and the protective substance removed from the aldehyde end of the molecule to produce a glucuronic acid compound. The synthesis of pure glucuronic acid was first successfully carried'out, so far as applicant is aware, by the process set forth 'in the above mentioned copending application. In that process, a glucose compound having a suitable substance coupled on the aldehyde end of the molecule is oxidized by nitrogen dioxide gas and the oxidized material hydrolyzed to free the glucuronic acid. The resulting product, which contained numerous impurities, isthen subjected to a rather elaborate extraction process. In view of the fact that on' oxidationthe glucose compound forms a heavy gummy-mass which is not readily penetrated by the nitrogen dioxide, the above mentioned process employed'the steps of suspending the material in chloroform in which was dissolved the nitrogen dioxide gas, with the result that the gas, on being released from solution, would force itself way up through the mass of material and eiiect the necessary oxidation. While the prior process was successful, it was long and costly and the yield of pure glucuronic acid was very low. In particular, a very extended periodof from seven to eight days was required to-complete the oxidation of the glucose compound, and thereafter elaborate extraction processes were required to produce small quantities of pure glucuronic acid or a salt thereof.

Accordingly, it is the general object of the present invention to improve, simplify, cheapen andincrease the speed and yield of the above described prior process of synthesizing glucuronic acid and its salts.

In" accordance with the present invention, substantial improvement in the process is realized bya combination of severalfeatures. In the first place} it ispreferred, although not necessary, to

subject to the oxidizing process a pure glucose compound having a protected aldehyde end; secondly, the speed of the oxidation process is greatly increased and its cost reduced by exposing an exceedingly thin film of dry powdered glucose compound directly to the nitrogen dioxide; and, third, the process of purifying the oxidized material is simplified and the yield increased by removing from the oxidized material all uncombined nitrogen compounds prior to the hydrolyzation' operation which is employed to remove the protecting substance on the aldehyde end of the molecule.

These three operations, alone, greatly simplify the process of purifying and extracting the glucuronic acid, and materially increase the yield.

In addition to the above, an even more-important feature of the invention resides in the step of removing combined nitrogen dioxide from the oxidized material before it is hydrolyzed. 'Ihisoperation, alone, is found to overcome most of the difiiculties which have been encountered in previous attempts to synthesize glucuronic acid, and it makes possible a yield of almost one hundred per cent of the theoretical yield of pure glucuronic acid with fewer extraction operations. It is now believed that most of the prior difiiculties which were encountered resulted from' the previously unrecognized fact that during the oxidation of a protected glucose compound, certain nitroglucose compounds are formed which seriously'reduced the yield and which introduce impurities that are very difiicult to remove.

The formation of these nitrogen compounds results because in oxidizing a mass of a glucose compound, such as methyl glucoside, with nitrogen dioxide, all of the glucose molecules do-not contact with the gas at one time, andhence the reaction progresses slowly through the mass. On each molecule, the carbon atoms tend to oxidize progressively, with the sixth carbon atom being the'first to oxidize and then others, probablythe second, third, fourth and fifth carbon atoms, in the order stated, the'first carbon atom being protected from oxidation, as previously indicated, and the'sixth carbon atom being the one which must be oxidized to produce glucuronic acid. When nitrogen dioxide is the oxidizing agent, the reaction occurs in two stages on each carbon atom:' first, a coupling of the nitrogen dioxide with'the carbon atom; and, second, a release. of the nitrogen. The object of the process is to oxi dize the sixth carbon atom Without affectinglthe others; but, due to the unavoidable oxidationroi some molecules in advance of others in the mass,

nitrogen dioxide couples with one or more of the intermediate carbon atoms on those molecules which are first oxidized at the sixth carbon, before the bulk of the material is oxidized at the sixth carbon. If the nitrogen dioxide which is thus coupled to an intermediate carbon atom is driven off as nitrous oxide, leaving an oxygen atom on the carbon, the chain breaks, thus reducing the yield and producing impurities which are difiicult to remove. The nitrogen on intermediate carbon atoms may be driven off some of the molecules as nitrous oxide during the oxidizing process if it is carried out too far, or in any case during the subsequent hydrolyzing operation, which occurs at a higher temperature. In addition, nitrogen dioxide may be driven off during the hydrolyzing operation and form nitric acid, which, at the higher temperatures then prevailing and in the presence of another mineral acid, will break the glucose chain. Not only does this cause a substantial reduction in yield and introduce a variety of undesirable glucose derivatives, but, if the nitrogen on intermediate carbons is not driven off, it remains in place and the final product, instead of being pure glucuronic acid, contains various nitroglucuronic acids. All of these difficulties are removed by stopping the oxidizing reaction before any appreciable per cent of the glucose molecules is broken and by then removing all combined nitrogen dioxide at low temperature, which will not break the nitrogen dioxide into oxygen and nitrous oxide.

The raw material for the process may constitute any protected glucose compound. Examples include ethyl glucoside, methyl glucoside, or polymerized glucose compounds, such as edible corn starch and glycogen. However, polymers give a lower yield and, therefore, the simpler nonpolymerized compounds are preferred. Because of its low cost and the ease with which it may be prepared in pure form, the preferred protected glucose compound is methyl glucoside, although, so far as the process, itself, is concerned, ethyl glucoside or glucosides having other protective alcohol ends for the aldehyde group are equally effective as starting materials. It will be understood that for such purposes, compounds of d. glucose (1. e., dextrose) are preferred.

The first step in the process involves the oxidation of the glucoside. This is accomplished by exposing the crystalline glucoside within a closed chamber to an atmosphere comprising a mixture of nitrogen dioxide and oxygen. The ratio of these two gases employed may be varied widely as desired, but one satisfactory proportion is two parts of nitrogen dioxide to one of oxygen. The oxygen is not essential to effect an oxidation of the glucoside, but its presence is highly advantageous for the reason that the reaction between the glucoside and nitrogen dioxide converts the nitrogen dioxide to nitrous oxide (acid). In the presence of oxygen, the nitrous oxide is immediately re-oxidized to nitrogen dioxide, thus preventing a loss of the relatively expensive nitrogen dioxide gas and at the same time preventing the accumulation of nitrous oxide in the oxidized material.

It is essential, in order tospeed the reaction, to maintain an intimate contact between the glucoside and the oxidizing gas, and this result may be achieved in several different ways. For example, the crystalline glucoside may be spread in a very thin layer on a supporting surface located within the gas chamber. This layer should preferably be in the order of one-half millimeter in thickness. The supporting surface may be a stationary tray of glass or stainless steel, or it may be a continuously moving stainless steel belt which carries the material through the reaction chamber. The oxidation process develops water, which progressively liquifies the material. Consequently, if desired, the supporting surface may be so inclined that the oxidized material will run off. In the latter case, a series of oppositely inclined supporting surfaces may be provided in the chamber so that the material which runs off one surface drips upon the next lower surface, and so on. Still another method of effecting an even more intimate contact is to drop finely divided crystalline material into an upwardly moving column of oxidizing gas. By properly adjusting the length of the column and the rate of flow of the gas, the descent of the non-oxidized material by gravity may be retarded for a sufiicient period to effect substantially complete oxidation before the glucose material, which will then be heavier, reaches a suitable supporting surface at the bottom of the column. If necessary, the oxidation may then be completed by permitting the material to run downwardly over a series of inclined surfaces in the bottom of the gas filled column, as mentioned above.

The speed of the oxidizing reaction varies with the density and the concentration of the nitrogen dioxide. The more concentrated and the denser the nitrogen dioxide, the faster is the reaction. The speed of the reaction also depends on the intimacy of the contact between th gas and gluCOse material. Since a reduction in the temperature of the gas increases its density, it will be apparent that either a decrease in temperature or an increase in the pressure within the chamber may be employed to increase the speed of the reaction. It is not absolutely essential that all of the glucose material be oxidized, since any residual unoxidized material is removed during the subsequent treatment. Therefore, the only effect of incomplete oxidation is to decrease the yield. It may be more economical, therefore, to reduce the yield in order to save time during the oxidizing operation. As a general guide to the employment of the process, it may be pointed out that with a layer of crystalline glucoside one-half millimeter thick supported on a horizontal plate in a closed chamber at room temperature and atmospheric pressure, with two parts of nitrogen dioxide and one part of oxygen, optimum yields were obtained in about eight hours, but no appreciable reduction in yield was noted until the exposure was extended beyond sixteen hours. Under these circumstances, the gas is preferably given a gentle circulation by cooling one side of the chamber and applying a mild heat to the opposite side. It will be apparent that the exposure time for optimum yields may be reduced by providing a more intimate contact between the nitrogen dioxide and the glucoside.

As soon as the material is oxidized, any combined nitrogen dioxide is then removed, as previously stated, by hydrolysis. This is accomplished by adding dilute sulphuric acid, ten cubic centimeters of five per cent sulphuric acid for each fifty grams of the original glucoside being adequate for this purpose. This operation should be carried out at a temperature not exceeding 40 C., in order to avoid breaking the glucose chain. The nitrogen dioxide which 18 thuscompletelyi removed from the-oxidized-:ma- ;;-terial' within a few minutes: either passes: oft-in the. form of. nitrogen dioxidegas. or; forms nitric lacidewith, the water; present.

The oxidized glucoside (i. e., methyl glucuronic ;a.cid,..ifqmethyl gluoosideiisithe, starting ,mate- .rial), together with the water, sulphuric acid, nitric.-.acid=and certain impurities, includingany .cellulosic compounds-which .may have resulted from a breakdown and/or condensation ofv the glucoside during oxidation are then dissolved in absolute. alcohol. .-A.-strongalcohol solution is required. Hence, if absolute-ethyl alcohol is .used,...the.amount added should be-a-pproximately .ten...times.the volume of the -water;added in the ,previous .step. .Absolute methyl alcohol in ..amounts from five to ten times the volume of water. may be used, and is preferred.

The..sulphuric. and nitric acidsarethen re- ..moved from the. alcohol solution. by additionof .an excess ofbarium carbonate. andprecipitation .of' the ba'rium..su1phate and. barium nitrate .-or

nitritefthm. formed. .Barium carbonate is-almost insoluble in alcoholbut nevertheless will progressively dissolve andneutralize the acids to produce the insoluble barium salts.

The solution may be maintainedat room temperature, but, if warmed to a temperature not above .50"

0.; the reactioniis speeded. One to three hours The arerequired to complete this reaction. barium carbonate willv not react appreciably with the methyl glucuronicacid in a strong alcohol solution. .The. precipitated barium-sulphate and and reduces the. product to .a syrup.

Since barium nitrate andbarium nitrite are soluble in water and the aboveprecipitationof barium nitrate and barium nitrite occurred in an alcohol-solution. containingsome water, there is a 'possibilitvthatat'this stage some of these barium salts remain inthe product. This is "checked'bydissolving a sample in Water and add- "ing sulphuric acid. "If any precipitateforms; the

batch'is againfdissolved in a strong alcohol solution. Since'at this stage less'water is present, all-of these barium-salts willprecipitate. The

material isthen again concentrated .to a syrup which .is principally .methyl glucuronic acid, the

only impuritiesbeing very small quantities of unoxidized methyl glucoside and cellulosic ,compounds resulting from the'breakdown of glucose chains.

The methyl glucuronic. acid, which is then entirely free of all nitrogen but may contain minor -amountsyofqother impurities, is then hydrolyzed with any suitable mineral acid. Sulphuric acid is preferred because it is less destructive to the glucose compound and its salts are readily removed. Hydrochloric acid is somewhat more de- .structiveiof the glucose compounds andnitric acid is even less desirable for the same reason, but both maybe used, ifdesired. The methyl glucuronicacid may .-be hydrolyzed .in acne per cent sulphuric acid solution at.an elevated tem- .hol.

1. the temperature;.jshould;be keptzas' low: as; possible without unduly prolonging the time: required to;complete;.the; operation. ;;Because.lofithermore :destructive.character;of hydrochlordcuandmitric zacids,=;even lower temperatures.:should :be .employedif they are'used.

gTheresulting solution contains pure glucuronic .acid and sulphuricacid. :Theacidsarethen com- .pletely zneutralizedzbyi. the, additionpf anexcess quantity .of barium; carbonate. .The; solution :.-is

"then chilled to precipitate the excess :bariumrcar- .bonate and the precipitate is removed by."fi1tra- .tion. .The filtrate-is concentrated byvacuumrdistillation; to the: consistency of -.a light syrup: and poured .into absolute alcohol. ,barium glucuronate, which is then :filtered-.-out :and. dried.

This precipitates in order to removethevbarium' from the'com- .pound, the barium glucuronate is. combinedsw-ith ..a calculated amount. of sulphuric acid solution slightly insufiicient in quantity to react with; the entire -quantity'. of barium glucuronate; present. :This forms barium sulphate, which, together-with .the residual-barium, glucuronate, may then .be

precipitated out. in absolute alcohol and filtered off. The remaining filtrateis an---alcohol solution of, pure glucuronic acid. This solution-may be subject tovacuumdistillation toecrystallize out the glucuronic, acid, .or,; if desired,.-,-may-= be neutralized with carbonates of sodium, potassium, calcium or, magnesium .to produce. the. corresponding glucuronate. ;These:may either-be re- ..tained in .a' vvatersolutionor crystallized'out by a vacuum distillation.

It will. be observed that several .of zthe-above describedoperations involve the precipitation of compounds in absolute alcohol. 'For this purpose, either ethyl or methyl alcohol i is preferred be- .cause of. their low. cost. and availability. .-However, comparable results =may...be,achieve d with any .monohydric 1 alcohol, such as ..propyl and .butyl. ,It is. not. essential that absolutealcohol be employed, but larger quantities of. dilutealcohols are required. to produce the same results and, therefore, absolute alcohols are preferred.

.At various stagesin the process, acids are .preferably neutralized bytheuse of barium carbonate. For this, purpose, barium carbonate is .pre-

'ferred because even excessquantities -wil1..not produce .an alkaline solution due to the..relative insolubility of the material. ,Th-isis-desirable since .an alkaline solutiontends vtobresik the glucose chain. Barium hydroxide may be employed in the place of barium carbonate, but ,in that case greatercare. is required to prevent. the formation of an alkaline solution. Sodium, .potassium and ammonium carbonates and hydroxides may likewise be employed, louttare .less ,desirable since the sodium, potassium .andammonium. ionsjdo not ,.precipitate as. readilyiualco- This reducesthe yield of-the-process. .ZCBJ- cium carbonate is subjectto the further defect that it will not completely neutralize the acids.

The abovedescribed process produces almost the full theoretical yieldof pure glucuronic acid from methyl glucoside, .as compared with yields of less thantenper cent with the. slower. and more complicated process of obtaining pureglucuronic acid disclosed in .applicantsprior.application, referred to. above. .If. the abovedescribed hydrolyzing step to remove combined nitrogen dioxide is omitted, yields in the order of fifty per cent of the theoretical can be obtained, provided the impure methyl gulcuronic acid, prior to removal of the methyl group, is converted to pure methyl barium glucuronate in the following manner:

The methyl glucuronic acid, after removal of the barium nitrate and ethyl (or methyl) nitrite and the vacuum distillation, is dissolved in water and an excess of barium carbonate is added to neutralize the methyl glucuronic acid and thus produce barium methyl glucuronate. The solution is concentrated by vacuum distillation at a temperature not to exceed C., to the consistency of a filterable syrup. This removes all of the alcohol. The material is then filtered to remove the excess barium carbonate, and its volume is further reduced by vacuum distillation to a syrup consistency. The syrup is then poured into absolute alcohol, which precipitates methyl barium glucuronate and barium salts of certain cellulosic condensation products which result from a breakdown of the glucose chain. The remaining impurities remain dissolved. The solution is filtered to remove the precipitates and the precipitates are dried and dissolved in cold water. Since only methyl barium glucuronate is soluble in cold water, all of the remaining impurities are removed by filtration, leaving a Water solution containing only methyl barium glucuronate, except that, as previously indicated, some of the material contains combined nitrogen dioxide.

The methyl barium glucuronate is then hydrolyzed and processed in the manner described above to produce glucuronic acid, the only difference being that in the hydrolyzing operation the barium precipitates as barium sulphate and is removed with the excess barium carbonate used to neutralize the acids. The resulting product is a mixture of pure glucuronic acid and nitroglucuronic acids, and the latter may be converted to pure glucuronic acid by hydrolyzing with sulphuric acid at low temperature, and the acids neutralized in alcohol and their salts removed by filtration and vacuum distillation in the manner employed with the methyl glucuronic acid in the preferred process.

It will also be understood that, if desired, the preferred process, first described above, may be modified by converting the impure methyl glucuronic acid to pure methyl barium glucuronate after the combined nitrogen dioxide has been removed and prior to the second hydrolyzing operation. This can be accomplished in the manner described above.

While several forms of the process are set forth herein, it will be apparent that other variations in the sequence and character of the steps may be indulged in without departing from the spirit of the invention or the scope of the appended claims.

What is claimed is:

1. The method of producing glucuronic acid, comprising first oxidizing a glucoside having a protected aldehyde group and a free primary alcohol group with nitrogen dioxide to form the corresponding derivative of glucuronic acid, extracting the oxidation products with alcohol and neutralizing nitric acid in the alcohol solution, recovering the alcohol soluble constituents from the solution and extracting the same with water, neutralizing said derivative of glucuronic acid in the aqueous solution and precipitating the salt thus formed in alcohol, extracting the substanfrom the residue with water, hydrolyzing the salt while in aqueous solution with a mineral acid, and removing the mineral acid and other impurities.

2. The method of producing glucuronic acid, comprising first oxidizing a glucoside having a protected aldehyde group and a free primary alcohol group with nitrogen dioxide to form the corresponding derivative of glucuronic acid, separating combined nitrogen dioxide from the oxidized glucose compound by hydrolysis at a temperature not exceeding 40 C., extracting the oxidation products with alcohol and neutralizing nitric acid in the alcohol solution, recovering the alcohol soluble constituents from the solution and extracting the same with water, neutralizing said derivative of glucuronic acid in the aqueous solution and precipitating the salt thus formed in alcohol, extracting the substantially pure salt of the glucuronic acid derivative from the residue with water, hydrolyzing the salt while in aqueous solution with a mineral acid, and removing the mineral acid and other impurities.

3. The method of producing glucuronic acid which includes subjecting a glucose compound having a free primary alcohol group and a protected aldehyde group to the action of a mixture of nitrogen dioxide and oxygen to convert the alcohol group to a carboxyl group, and separating glucuronic acid in solution by hydrolysis at a temperature not exceeding about C.

4. The method of producing a glucuronic acid compound which includes exposing crystalline methyl glucoside in a finely divided state to an atmosphere of oxygen and nitrogen dioxide at a temperature not exceeding 50 C. and thereafter separating glucuronic acid by hydrolysis at a temperature not exceeding about 90 C.

5. The method of producing a glucuronic acid compound which includes dropping finely divided crystalline methyl glucoside by gravity into an upwardly moving column of a mixture of nitrogen dioxide and oxygen and thereafter separating glucuronic acid by hydrolysis at a temperature not exceeding about 90 C.

6. In the method of producing glucuronic acid which includes subjecting a glucose compound having a free primary alcohol group and a protected aldehyde group to the action of nitrogen dioxide to convert the alcohol group to a carboxyl group and thereafter separating glucuronic acid in solution by hydrolysis, the step of separating combined nitrogen dioxide from the oxidized glucose compound by hydrolysis at a temperature not exceeding 40 C.

EARL A. PETERMAN.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,232,990 Yackel et al. Feb. 25, 1941 2,379,917 Mark et a1 July 10, 1945 OTHER REFERENCES Maurer et al., Ber. Deut. Chem. Gessell, vol. 75, pages 1490-1491 (1942). 

3. THE METHOD OF PRODUCING GLUCURONIC ACID WHICH INCLUDES SUBJECTING A GLUCOSE COMPOUND HAVING A FREE PRIMARY ALCOHOL GROUP AND A PROTECTED ALDEHYDE GROUP TO THE ACTION OF A MIXTURE OF NITROGEN DIOXIDE AND OXYGEN TO CONVERT THE ALCOHOL GROUP TO A CARBOXYL GROUP, AND SEPARATING GLUCURONIC ACID IN SOLUTION BY HYDROLYSIS AT A TEMPERATURE NOT EXCEEDING ABOUT 90*C. 